Efficacy of a 0.5% Propolis -0.9% Pomegranate Buccal Spray Treatment Compared with 2% Miconazole Gel for Denture Stomatitis Treatment in Elderly Patients: a Randomized Clinical Trial

Statement of the Problem: Natural products have attracted interest as an alternative to synthetic medi-cations for the treatment of oral diseases due to their efficacy and safety. Propolis and pomegranate extracts have both demonstrated efficacy for the treatment of denture stomatitis. However, use of the two compounds together has not been tested for this purpose. Purpose: A comparison was made of the efficacy of a commercially available propolis-pomegranate buccal spray formulation for the treatment of denture stomatitis, compared with miconazole gel, based on stomatitis lesions and Candida spp. concentrations in mouth rinses. Materials and Method: This was an experimental study, characterized as an open-label, parallel two-armed, non-inferiority randomized clinical trial. Forty elderly adults aged < 60 years with denture stoma-titis were randomly allocated to two groups. The patients applied a buccal spray containing 0.5% propo-lis and 0.9% pomegranate extracts or 2% miconazole gel, a standard treatment recommended in Brazil, to the inner surface of their dentures three times a day for 14 days. They were examined at days 1, 7, 14 and stomatitis lesions were categorized according to Newton’s score. Mouth rinses were made with saline solution at days 1 and 14 and then assessed for Candida spp. Results: Both treatments reduced the Newton’s score, with clinical cure rates of 75 and 40% for the miconazole and propolis-pomegranate groups, respectively. The Candida concentrations in the mouth rinse decreased significantly only in the miconazole group. Conclusion: The propolis-pomegranate spray was less effective than the miconazole treatment. Howev-er, clinical improvement was also observed in patients treated with the propolis-pomegranate buccal spray.


Introduction
Denture stomatitis (DS) is generally caused by Candida spp. [1][2]; it is characterized by erythematous lesions with variable intensity and extension, often affecting the palatal mucosa of denture wearers. The treatment of choice consists of topical antifungals, such as nystatin or miconazole and proper denture hygiene, as well as removal of the denture at night [3]. However, there are cases of therapeutic failure and of rapid recurrence after treatment, especially in the absence of proper denture hygiene [3]. Moreover, the increase in resistance of Candida spp. yeasts to commercially available antifun-ing the potential antimicrobial activity of P. granatum.
Another study showed concentration-dependent activity against Candida spp. of an alcoholic extract of P. granatum peel in salivary samples collected from patients with oral candidiasis [25]. A clinical trial revealed clinical and microbiological efficacy of a laboratoryproduced P. granatum gel in the treatment of patients with DS [25].

Study design
This was an open-label, parallel two-arm, non-inferiority randomized clinical trial with a miconazole control. years, wearer of removable partial or complete denture with palatal coverage, and diagnosis of DS. Exclusion criteria were treatments with any kind of antifungal product, antibiotic or anti-inflammatory drug for two months prior to recruitment; dementia or cognitive deficit were also included as exclusion criteria. Demographic and clinical history data were collected via anamnesis at day 0 (T0) of the study. The participants answered questions about denture age, frequency of daily oral hygiene, and habit of wearing the denture while sleeping. DS was diagnosed via intra-oral examination by a trained dental surgeon at T0, T7 and T14.
Newton's classification [27] was used for the lesions, categorized as I (localized inflammation or petechiae), II (diffuse erythema involving all or part of the hard palate covered by the denture), and III (erythema associated with papillary hyperplasia in the area covered by the denture). All oral lesions were photographed and reviewed by a second blinded evaluator. In case of disagreement between the evaluators, the photos were reviewed and a reclassification was made.

Randomization and allocation
Participants were randomly assigned to study groups according to a random number table produced with http://www.graphpad.com/quickcalcs, GraphPad Software, Inc. A second researcher generated the random allocation sequence, and the principal researcher allocated the participants into the groups (MIC-miconazole group; PP-propolis-pomegranate group) based on this random sequence.

Interventions
Participants applied 2% miconazole oral gel (Daktarin ® Gel Oral, Janssen-Cilag Pharmaceutics) or a waterbased spray containing 0.5% propolis extract and 0.9% originating from Baccharis dracunculifolia [10]. Participants were instructed to perform prosthesis hygiene by brushing with dentifrice and to sprinkle 0.12% chlorhexidine digluconate solution on the inner surface of the denture to remove excess chlorhexidine, and to apply a thin layer of 2% miconazole oral gel or to spray Apiromã ® on this surface, three times a day for 14 days.
All participants received instructions on oral hygiene, denture hygiene maintenance, use of the products, and removal of dentures for nighttime sleep. The dentures were stored at night in water. Participants received a tube of miconazole or Apiromã ® buccal spray with preweighed amounts, 0.12% chlorhexidine solution, and writ-ten instructions on the use of the products.
For microbial load analysis, participants rinsed their mouths with 20 mL of sterile 0.9% sodium chloride at T0 and T14 in the dental clinic. The rinse samples were collected into Falcon tubes, kept on ice, and processed up to one hour after collection. They were centrifuged for 15 minutes at 25°C at 3,000xg; the supernatant was discarded, and the pellets were resuspended in 1mL of 0.9% sodium chloride solution, and then the suspensions were diluted 10:1 successively, to provide 10 -1 to

Outcomes
Clinical cure (rate of DS resolution), defined as absence of lesions, was the main outcome. Secondary outcomes were fungal load reduction (greater than 90% reduction of CFU/mL counts), rate of adverse events, and product acceptance.

Sample size
Sample size was estimated as described by Chow et al. [29] for parallel two-arm studies with dichotomous outcomes. Based on previous data [14], a non-inferiority margin of 0.15 (15% variation between treatments is considered clinically nonsignificant), 70% response rate for miconazole, 70% response rate for Apiromã ® , 5% significance level, and 80% power of the study were adopted. Accordingly, a sample size of 18 individuals per arm was estimated. Given an estimated 10% loss to follow-up, 20 participants per arm were recruited.

Statistical analysis
The data were reported as means or medians, with the respective measures of variability. The Mann-Whitney test was used to analyze the magnitude of the effect (clinical efficacy) and product acceptance. A noninferiority interval was calculated from the data observed in the standard treatment group, with a non-inferiority margin of 15%. The chi-square test was used for comparison of cure rates and rates of adverse events.
GraphPad Prism v 7.0 was used for statistical analyses.
p Values <0.05 were considered statistically significant.   The clinical cure rate was 75% for the MIC group and 40% for the PP group (Table 2); consequently, the hypothesis of non-inferiority of Apiromã ® compared to miconazole was rejected. Also, CFU/mL in 20ml buccal wash decreased in the MIC group (p< 0.0001), but not in the PP group (p= 0.70) (Figure 3).

Results
According to the hedonic scale data, product acceptance was similar in the two groups (p= 0.72). The median was 5 (4.2-6.0) for the MIC group and 5.5 (4.2-6.0) for the PP group (Table 3).

Discussion
An evaluation was made of a commercial formula (Api- Newton' score (T0)** 1.8(±0.8) 1.9(±0.8) 0.70 a * Patients self-declared themselves as having white skin or not. **Newton' score: I (localized inflammation or petechiae), II (diffuse erythema involving the total or partial area of the hard palate covered by the denture) and III (erythema associated with papillary hyperplasia in the area covered by the denture).    This product did not reduce the fungal load significantly, demonstrating a lack of antifungal efficacy. The clinical trial performed by Pina et al. [14] in which they used a mucoadhesive gel containing the same propolis extract used in this study (EPP-AF ® ), but at a higher concentration (2%), had a higher clinical cure rate (70%) for DS, but also did not have antifungal efficacy.
These findings could be explained by the effect of propolis on C. albicans dimorphism, provoking the transition of more pathogenic forms, these being pseudohyphae and hyphae to yeasts, by affecting the immune system [30]. This phenotypic change from hyphae and pseudohyphae to yeasts is induced by propolis without quantitative reduction in CFU/mL [13]. These findings lead to the conclusions that propolis does not have an antifungal effect at the tested doses (1.5-2.0mg of propolis per application of the buccal spray), and that its anti-inflammatory and healing properties predominate over its antifungal activity [14].
Another study demonstrated clinical and microbiological efficacy of green propolis when used as 2.5% gel and 24% mouth rinse, applied four times a day for seven days for DS [15], a dosage much higher than that used in our current study (0.5%), indicating that the posology should be adjusted.

Conclusion
This randomized controlled trial demonstrated that the 0.5% propolis and 0.9% pomegranate extract buccal spray product Apiromã ® can help control stomatitis in denture users, though it is less efficient than 2% miconazole gel, which is commonly recommended and used.

Data Availability
All data of this study are available from the corresponding author upon reasonable request.